Waveguides for performing enzymatic reactions

ABSTRACT

The present invention is directed to a method and an apparatus for analysis of an analyte. The method involves providing a zero-mode waveguide which includes a cladding surrounding a core where the cladding is configured to preclude propagation of electromagnetic energy of a frequency less than a cutoff frequency longitudinally through the core of the zero-mode waveguide. The analyte is positioned in the core of the zero-mode waveguide and is then subjected, in the core of the zero-mode wave guide, to activating electromagnetic radiation of a frequency less than the cut-off frequency under conditions effective to permit analysis of the analyte in an effective observation volume which is more compact than if the analysis were carried out in the absence of the zero-mode waveguide.

CROSS-REFERENCES

This application is a continuation application of Ser. No. 11/313,971,now U.S. Pat. No. 7,181,122, filed Dec. 20, 2005 which is a continuationapplication of Ser. No. 11/151,807, now U.S. Pat. No. 7,013,054, filedJun. 13, 2005, which is a continuation application of Ser. No.10/259,268, now U.S. Pat. No. 6,917,726, filed Sep. 27, 2002, whichclaims priority to U.S. Provisional Patent Application No. 60/325,280,filed Sep. 27, 2001, which is related to U.S. patent application Ser.No. 09/572,530, all of which are hereby incorporated by reference intheir entirety.

This invention was made with funds provided by the U.S. Government underNational Science Foundation Grant No. BIR8800278, and NationalInstitutes of Health Grant No. P412RR04224-11, and Department of EnergyGrant No. 066898-0003891. The U.S. Government may have certain rights inthis invention.

FIELD OF THE INVENTION

The present invention is directed to zero-mode metal clad waveguides forperforming spectroscopy with confined effective observation volumes.

BACKGROUND OF THE INVENTION

Observing and understanding the activity of a single molecule, such asan enzyme, is critical to understanding the dynamics of many importantbiochemical processes, such as catalysis, signal transduction, and generegulation. Many biochemical reactions require micromolar ligandconcentrations. In order to perform spectroscopy on one or a fewmolecules at such high concentrations, it is necessary to limit the sizeof the effective observation volume.

Previous attempts at sub-diffraction limited spectroscopy have includedthe utilization of near-field apertures. These implementations typicallyinvolve an optical fiber that has been tapered to a sub-wavelength pointand coated with a metal such as aluminum. A subwavelength aperture isformed in the metal at the end of the fiber. Excitation light is sentdown the fiber towards the aperture, and the elements to be studied arepresent outside the fiber and in close proximity to the aperture. Thesubwavelength nature of the aperture results in a light diffractionpattern that includes evanescent modes. These modes rapidly decay withdistance from the aperture, thus effectively confining the volume ofillumination. Only a very small percentage of light sent down the fibermakes it through the near-field aperture to the illumination region,making the prior art very inefficient.

Additionally, the spectroscopic signal from the analyte is bestcollected externally by an additional apparatus, such as a microscopeobjective, since the efficiency of collection by the near-field fiber isvery low.

The present invention is directed to overcoming these deficiencies inthe prior art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for analysis of ananalyte. The method involves providing a zero-mode waveguide whichincludes a cladding surrounding a core, where the cladding is configuredto preclude propagation of electromagnetic energy of a frequency lessthan a cutoff frequency longitudinally through the core of the zero-modewaveguide. The analyte is positioned in the core of the zero-modewaveguide and is subjected, in the core of the zero-mode waveguide, toactivating electromagnetic radiation of a frequency less than the cutofffrequency under conditions effective to permit analysis of the analytein a volume that is more compact than if the analysis were carried outin the absence of the zero-mode waveguide.

The apparatus of the present invention is used for analysis of ananalyte. This includes a zero-mode waveguide having a claddingsurrounding a core, where the cladding is configured to precludepropagation of electromagnetic energy of a frequency less than a cutofffrequency longitudinally through the core of the zero-mode waveguide. Inaddition, this apparatus has a source of electromagnetic radiationpositioned relative to the zero-mode waveguide to direct electromagneticradiation of a frequency less than the cutoff frequency into the coreunder conditions effective to permit analysis of an analyte in aneffective observation volume which is more compact than if the analysiswere carried out in the absence of the zero-mode waveguide.

In one embodiment of the present invention, the apparatus includes asuperstructure contacted with the opaque film that serves to facilitatethe use of a single chip with several different samples to be analyzed.In this embodiment, the zero-mode waveguides are positioned in an arraysuch that several identical devices are spaced at equal intervals acrossthe surface of the chip. The superstructure serves to isolate eachdevice on the chip from all of the rest, allowing an individual deviceon a chip to be used with a particular sample without contaminating therest of the devices on the chip.

In another embodiment of the present invention, a differentsuperstructure is applied to a chip containing an array of zero-modewaveguide devices to facilitate the delivery of a small, accuratelymetered quantity of sample to each device on the chip. In thisembodiment the superstructure contains microfluidic channels positionedto allow sample introduced at one or more input ports to be delivered tofluid cavities positioned over each of the zero-mode waveguide devices.The microfluidic portions of the superstructure can be used simply toconvey and measure the sample, or more sophisticated operations such ascapillary electrophoresis can be performed on the sample before itreaches the zero-mode waveguide device for optical analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a metal-clad zero-mode waveguide withlateral dimensions, d, less than half the wavelength of illuminatinglight, in accordance with the present invention.

FIGS. 2 is a cross-sectional view of one embodiment of a zero-modewaveguide in accordance with the present invention.

FIGS. 3A, 3B, 3C and 3D are a series of cross-sectional views, depictingone embodiment for the preparation of a zero-mode waveguide inaccordance with the present invention.

FIGS. 4A, 4B, and 4C are a series of cross-sectional views, depictinganother embodiment for the preparation of a zero-mode waveguide inaccordance with the present invention.

FIGS. 5A, 5B, 5C, 5D and 5E are a series of cross-sectional views,depicting a further embodiment for the preparation of a zero-modewaveguide in accordance with the present invention.

FIGS. 6A, 6B, 6C and 6D are a series of cross-sectional views, depictinganother embodiment for the preparation of a zero-mode waveguide inaccordance with the present invention.

FIGS. 7A, 7B, 7C and 7D are a series of cross-sectional views, depictinga further embodiment for the preparation of a zero-mode waveguide inaccordance with the present invention.

FIGS. 8A and 8B show an alternative embodiment of a zero-mode waveguidein accordance with the present invention where the waveguide is formedfrom an optical fiber and an enlargement of the tip of the zero-modewaveguide.

FIGS. 9A and 9B show a system utilizing a zero-mode waveguide inaccordance with the present invention.

FIG. 10 is a scanning electron micrograph showing a top view of azero-mode waveguide prepared in accordance with the present invention.

FIG. 11 is a contour plot of the logarithm of the intensity distributionin a 50 nm diameter cylindrical waveguide viewed from the side at thediameter. The heavy lines mark the borders of the waveguide, and thecalculation is for 500 nm light in a water-filled, aluminum-cladwaveguide.

FIG. 12 is a graph of the intensity squared as a function of depth intocylindrical waveguides of various diameters for 500 nm light.

FIG. 13 is a graph of the effective observation volume in cylindricalwaveguides as a function of waveguide diameter for 500 nm light.

FIG. 14 is a drawing of the chemical structure of the dye BODIPY 515pyrophosphate (Molecular Probes, Eugene, Oreg.).

FIG. 15 is a graph fitting fluctuation correlation spectroscopy model todata for waveguides of various diameters.

FIG. 16 is a cross-section of a zero-mode waveguide in accordance withthe present invention used for observation of enzymatic activity.

FIG. 17 shows fluorescence correlation spectroscopy (“FCS”) curves froma waveguide before, during, and after use of a polymerase.

FIG. 18 shows a time trace of fluorescence during use of a polymerase.

FIG. 19 is a cross-sectional view of a chip containing zero-modewaveguide devices and superstructure for isolating individual zero-modewaveguide devices in an array from one another to facilitate independentanalysis of several samples on a single chip.

FIG. 20 is a perspective view of the device shown in FIG. 19.

FIG. 21 is a cross-sectional view of a chip containing zero-modewaveguide devices and superstructure for delivering aliquots of a singlesample to several zero-mode waveguide devices on a single chip by way ofthe same sample inlet port.

FIG. 22 is a plan view of the device depicted in FIG. 21.

FIG. 23 is a perspective view of a zero-mode waveguide constructed inconjunction with a fluid channel to allow analysis of analytes in thechannel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for analysis of ananalyte. The method involves providing a zero-mode waveguide whichincludes a cladding surrounding (i.e. partially or fully) a core, wherethe core is configured to preclude propagation of electromagnetic energyof a frequency less than a cutoff frequency longitudinally through thezero-mode waveguide. The cutoff frequency is defined as the frequencybelow which the waveguide is incapable of propagating electromagneticenergy along the waveguide under the illumination geometry used. In oneembodiment, the core is sufficiently small to preclude the propagationof electromagnetic energy of frequency below the cutoff frequency underany illumination geometry. A further embodiment uses a waveguide that iscapable of supporting one or more longitudinally propagation modes ofelectromagnetic energy. In this embodiment, a special illuminationgeometry is used such that minimal or no energy is coupled into thepropagating modes of the waveguide. The analyte is positioned in thecore of the zero-mode waveguide and is subjected, in the core of thezero-mode waveguide, to activating electromagnetic radiation of afrequency less than the cutoff frequency under conditions effective topermit analysis of the analyte in an effective volume which is morecompact than if the analysis were carried out in the absence of thezero-mode waveguide.

The apparatus of the present invention is used for analysis of ananalyte. This includes a zero-mode waveguide having a claddingsurrounding a core, where the core is configured to preclude propagationof electromagnetic energy of a frequency less than a cutoff frequencylongitudinally through the zero-mode waveguide. In addition, thisapparatus has a source of electromagnetic radiation positioned relativeto the zero-mode waveguide to direct electromagnetic radiation of afrequency less than the cutoff frequency into the core under conditionseffective to permit analysis of an aralyte in an effective observationvolume which is more compact than if the analysis were carried out inthe absence of the zero-mode waveguide.

The present invention relates to zero-mode waveguides and their use toconfine the effective observation volumes that are smaller than thenormal diffraction limit. As shown in FIG. 1, excitation radiation(e.g., incident light) L enters waveguide 2, having core 3 and cladding5, at entrance pupil 4. The waveguide comprises an internal volume withlateral dimensions (d) smaller than half the wavelength (λ) of theexcitation light. This internal volume (i.e. the core) is comprised of amaterial that includes, or is capable of including, one or more elementsof the material on which one or more forms of spectroscopy are to beperformed. The volume external to the internal volume in the lateraldirections is the cladding which is composed of a metal or metal-likematerial. The end of the waveguide opposite entrance pupil 4 is the exitpupil 6. The excitation power within the waveguide decays along thelength of the guide. The waveguide is long enough so that theobservation volume is predominantly confined to the region internal tothe waveguide. As a result, signal collected from extraneous elements isgreatly reduced.

FIG. 2 is a cross-sectional view of one embodiment of a zero-modewaveguide in accordance with the present invention. This waveguide 2 iscomprised of holes 8, which function as the core, in metal film 10,which functions as the cladding surrounding the core, on glass substrate12. A solution of analyte is placed above and inside the waveguides.Illumination L is from below the zero-mode waveguide, and thespectroscopic signal from the analyte is detected through the glasssubstrate. The waveguide of FIG. 2 is preferably prepared by providing aglass or fused silica cover slip with an aluminum film on it. Chromiumand other metals with small skin depth at the frequency of illuminationused are also suitable for use in the zero-mode waveguide of the presentinvention. Holes 8 can be formed in the film by electron beamlithography. See Example 1, infra.

Other embodiments of the zero-mode waveguide of the present inventioncan be made by a number of techniques.

In another embodiment, as shown in FIGS. 3A, B, C and D, electroplatingis used to progressively restrict larger holes in metal patterned usinga low resolution patterning technique, such as photolithography.Electroplating will plate new metal only on those areas alreadymetalized, so it will not deposit metal on the bare floors of the holes.However, it will deposit on the inside edges of the holes, so the holeswill grow smaller in diameter as the film grows thicker. By choosing theappropriate time to discontinue deposition, the holes can be madesufficiently small to act as zero-mode waveguides. To fabricate thesestructures, a film of an appropriate priming metal 20, such as gold, isdeposited on transparent (e.g., glass) substrate 22 (FIG. 3A). A layerof photoresist is then applied over the layer of priming metal (FIG.3B). A cost-effective lower-resolution lithography system, such as 248nm optical lithography, commonly known to practitioners in the art ofoptical lithography, produces holes in a photoresist mask layer 24 assmall as 150 nm in diameter (FIG. 3B). This pattern of holes 26 is thentransferred from layer 24 to the metal priming layer 20 (FIG. 3C) by wetetching, reactive ion etching, or ion milling—all techniques commonlyknown in the art. After removal of excess resist, a cladding material28, such as chromium, is then electroplated onto priming layer 20 (FIG.3D). The electroplating process will deposit cladding on the metalprimed surfaces 20 but not the bare glass surfaces 22 located at holes26. Because of this, the radius of the holes will shrink a distanceequal to the thickness of the plated film. By appropriate selection ofthe plating time, or by optical feedback control, the holes can beconstricted to much smaller dimensions. Holes as small as 1 nm can beconsistently fabricated using related techniques. See Kang et al.,“Investigations of Potential—Dependent Fluxes of Ionic Permeates in GoldNanotubule Membranes Prepared Via the Template Method,” Langmuir17(9):2753-59 (2001) (“Kang”), which is hereby incorporated byreference. After electroplating, the device is ready for use.

In another embodiment for preparation of zero-mode waveguides inaccordance with the present invention, as shown in FIGS. 4A, B and C, amembrane filter (commercially available under trade names, such asNUCLEOPORE membranes (Whatman, Clifton, N.J.), and TRACK ETCH membranes(Whatman, Clifton, N.J.)) is used in place of an electron beam resist asthe etch mask In techniques commonly known in the art of filterfabrication, free-standing films of polymer (usually polycarbonate) aresubjected to bombardment by energetic ionized nuclei, generated byeither a Van De Graff generator or a nuclear reactor. These fragmentspenetrate the entire thickness of the membrane and cause chemicalchanges to the polymer in the vicinity of the trajectory of theenergetic nucleus. These chemical changes induce a difference insolubility of the polymer in an etchant, commonly potassium hydroxide.The films are bathed for some duration in the etchant, causing the filmto dissolve in the vicinity of the nuclear trajectories in the membrane.These holes can be quite small (as small as 15 nm) and quite consistentin size (i.e., the variability is less than 4 nm). Film 30 fabricated inthis manner is directly applied to thin metal film 34 which is depositedon transparent substrate 32 (FIG. 4A). The pattern of pores 36 inpolymer film 30 is then transferred to the metal film by any of a numberof techniques (FIG. 4B), including reactive ion etching, wet chemicaletching, and ion milling. The remaining polymer film 30 is then removed(FIG. 4C) with a solvent suitable to dissolve the polymer (such astoluene or xylenes in the case of polycarbonate) or an oxygen plasmaclean process (known to those in the art of plasma processing).

In another embodiment for preparation of zero-mode waveguides inaccordance with the present invention, as shown in FIGS. 5A, B, C, D andE, a thin film of a suitable polymer material is deposited on a metalfilm and exposed to bombardment by high-energy ions and etched in amanner very similar to the etching techniques discussed above. In thisembodiment, cladding material (e.g., chromium, aluminum, or gold) 40 isevaporated onto transparent (e.g. glass) substrate 42 (FIG. 5A) and thinpolymer film 44 is spin-cast (FIG. 5B) directly onto the metal surface(as opposed to a free-standing membrane as in the embodiment of FIGS.4A-C). The entire substrate with the metal and polymer film is thensubjected to ion bombardment, as in the embodiment of FIGS. 4A-C, andthe polymer film is developed with a solvent which will dissolve zonesnear the trajectory of an energetic nucleus (FIG. 5C), but not theunaltered film. The pattern of holes 46 which is thus created in thethin film of polymer 44 is then transferred into metal layer 42 usingreactive ion etching, wet chemical etching, or ion milling (FIG. 5D).After pattern transfer, the remains of the polymer film are removed (asabove) with a suitable stripping solvent or an oxygen plasma (FIG. 5E).

In another embodiment for preparation of zero-mode waveguides inaccordance with the present invention, as shown in FIGS. 6A, B, C and D,the zero-mode waveguides are fabricated directly in etched membrane 50(FIG. 6A). Following the methods of Kang, a layer of gold 54 isdeposited on all surfaces of a polycarbonate film, including thecylindrical interiors of pores 52 (FIG. 6B). This gold film is used as apriming layer to electroplate a cladding layer material 56, such aschromium (FIG. 6C). The membrane filters are commercially available withpores in a wide variety of dimensions. In one embodiment, 100 nm poresare primed with 5 nm of gold and then plated with 30 nm of chromium,leaving a 30 nm interior waveguide core with a 30 nm cladding layer ofchromium. These structures can be optionally immobilized on atransparent (e.g. glass) substrate 58 and optionally thinned by any ofthe material removal techniques well-known to those in the art of thinfilm processing (FIG. 6D).

In another embodiment for the preparation of zero-mode waveguides inaccordance with the present invention, as shown in FIGS. 7A, B, C and D,cladding material lift-off is carried out using an opposite tone film orpattern. This embodiment involves first applying polysilicon-layer 72over light transmitting (e.g., fused silica) substrate 70, as shown inFIG. 7A. Polysilicon pillars 72A are then formed from polysilicon layer72 using conventional photolithography, as shown in FIG. 7B. Pillars 72Aare reduced in size by baking in an oxygen atmosphere followed bytreatment with an etchant, such as hydrofluoric acid. As shown in FIG.7C, substrate 70 and pillars 72A are then electroplated with metal layer74 and 74A, respectively. Gold is a suitable metal layer for suchpurposes. Pillars 72A and metal 5 layer 74A are then removed byconventional techniques, leaving behind metal layer 74 with holes 76over substrate 70, as shown in FIG. 7D.

In another embodiment for preparation of zero-mode waveguides inaccordance with the present invention, as shown in FIGS. 8A and B, afiber tip 80, which is similar to a near-field scanning opticalmicroscope tip is constructed so that it terminates with a zero-modewaveguide where the tip allows entrance of analyte material into theinterior of the zero-mode waveguide. This embodiment of the presentinvention is prepared by heating the end of an optical waveguide fiber82 and drawing it so that its diameter narrows at the heated end. Theresulting tapered tip is coated with zero-mode cladding material 84. Thesilica of fiber 82 is then etched (e.g., with hydrofluoric acid) a smalldistance to form hole 86 which serves as the core for the zero-modewaveguide.

The use of zero-mode waveguides, in accordance with the presentinvention, to analyze analytes is shown in FIG. 9B.

Surface 102 with metallic layer 116 applied over surface 102 with smallholes 118 etched into opaque layer 116 represents one embodiment of thepresent invention. When illuminated from below, the light cannotpenetrate fully through the holes into reagent solution R, because thediameter of holes 118 is smaller than one half of the light'swavelength. As shown in FIG. 9B, the material undergoing spectroscopicanalysis is positioned in hole 118 and is illuminated from below.Because the effective volume of observation is very small, signals dueto extraneous material in the vicinity will be reduced.

The system for carrying out analysis of analytes in accordance with thepresent invention is shown in FIG. 9A. Illumination source 110 (e.g., alaser) directs excitation radiation by way of dichroic beam splitter 108through lens 106 and surface 102 to the target material. This excitesthe material with the resulting emitted radiation passing back throughsurface 102 and lens 106. Dichroic beam splitter 108 allows passage ofthe emitted radiation to detector 112 which identifies the type ofemission. The detected emission information is then directed to computer114 where the material corresponding to the emission is identified andits identity stored. The radiation being emitted can be in the form offluorescence, Raman scattered light, Hyper-Rayleigh scattered light,luminescence, Hyper-Raman scattered light, or phosphorescent light.

The zero-mode waveguide of the present invention can be used to analyzea variety of analytes. Amongst these are biomolecules, such as proteinsand nucleic acids.

The zero-mode waveguide of the present invention can be used forsequencing nucleic acid molecules, as fully described in U.S. patentapplication Ser. No. 09/572,530, which is hereby incorporated byreference. This method involves providing a complex of a nucleic acidpolymerizing enzyme and a target nucleic acid molecule oriented withrespect to each other in a position suitable to add a nucleotide analogat an active site complementary to the target nucleic acid. A pluralityof types of nucleotide analogs are provided proximate to the activesite, where each type of nucleotide analog is complementary to adifferent nucleotide in the target nucleic acid, leaving the addednucleotide analog ready for subsequent addition of nucleotide analogs.The nucleotide analog added at the active site as a result of thepolymerizing step is identified. The steps of providing a plurality ofnucleotide analogs, polymerizing, and identifying are repeated so thatthe sequence of the target nucleic acid is determined. The zero-modewaveguide of the present invention is used to carry out the step ofidentifying the nucleotide analog added to the target nucleic acid.

The zero-mode waveguide of the present invention can also be used toanalyze other enzymatic reactions, including haplotyping with DNApolymerase, enzymatic reactions with RNA polymerase or helicase/primase,and analysis of ribosomes, spliceosomes, transcription complexes,chaperon proteins, protein folding, virus particle assembly, catalyticor non-catalytic antibodies, ribozymes, proteins involved in nucleicacid recombination, exonucleases, endonucleases, inorganic catalysts,and detection of viruses or other small pathogens.

The zero-mode waveguide of the present invention can be used to analyzeone or more analytes, either sequentially or simultaneously within thewaveguide.

EXAMPLES Example 1 Fabrication of Zero-Mode Waveguide Array

Arrays of zero-mode waveguides were manufactured as small holes in a 50nm thick film of aluminum on a glass or fused silica coverslip. Thesteps for the fabrication of the devices are as follows. First, thecover glasses were cleaned with a solution of one part ammoniumhydroxide, one part hydrogen peroxide, and six parts water at 70° C. Thecoverglasses were immersed in this solution for 10 minutes, and thenrinsed in a overflowing bath of deionized water for 10 minutes. Thesamples were dried with compressed dry nitrogen and then subjected tooxygen plasma for 3 minutes. The cleaned cover glasses were then coatedwith 50 nm of aluminum by thermal evaporation. An electron beamlithography resist, ZEP-7000A, was spun onto the cover glasses for 60seconds at 3000 RPM. Excess solvent was driven from the films by bakingon a temperature-controlled hotplate for 30 minutes at 170° C. Thisprocess yields a film approximately 300 nm thick. The films were thenmounted for exposure in an electron-beam lithography system. Electronbeam exposure was performed in a pattern of dots separated by 5micrometers (for optical isolation during use). A range of doses can beused to generate a gradation of hole sizes on a single coverglass forstudies where variable hole size is useful. The latent pattern was thendeveloped using a solution of xylenes at room temperature for 3 minutes.The development was stopped with a rinse of isopropanol, followedimmediately by drying with compressed dry nitrogen. The developedpattern was then transferred to the aluminum layer by reactive ionetching using an aluminum etch recipe: 20 sccm Cl₂, 40 sccm BCl₃, and 2sccm H₂. The pressure was maintained at 20 mT, and the radio-frequencypower was controlled by feedback to hold the sample bias potential at400 V. The etch proceeded for approximately 1 minute and 20 seconds.Immediately after removal from the etch chamber, the samples were rinsedin deionized water to remove residual chlorine radicals which candegrade the structures on exposure to moisture in the air. The remainingresist was exposed to Short-wavelength ultraviolet radiation to exposeit, and the exposed resist was removed with another developer for thisfilm: methyl isobutyl ketone (“MIBK”). Again, the samples were rinsed inisopropanol and blown dry with compressed dry nitrogen. The final stepbefore use was to subject them to an oxygen plasma to harden thealuminum native oxide and remove any organic residue from processing. Atotal of 3 minutes of exposure at 1000 watts was used, but theradio-frequency power was turned off and on to keep the substratetemperature below 120° C. to prevent damage to the aluminum film.

FIG. 10 is a scanning electron micrograph showing a top view of thezero-mode waveguide made by the process of this example.

Example 2 Intensity Evaluation with Zero-Mode Waveguide Array

Simulations of the electric field inside zero-mode waveguides wereperformed using a commercial finite-element time-domain Maxwell'sequation solver (EMFlex, from Weidlinger Associates, New York, N.Y.).Models were run for right, circular cylindrical waveguides of water in50 nm thick aluminum films on a glass substrate (index of refraction1.5). The entire region above the aluminum and inside the waveguides wasassumed to be filled with water, and the illumination was, in all cases,normally incident circularly polarized plane waves of light at 6×10¹⁴ Hz(corresponding to a vacuum wavelength of 500 nm). The entire region ofthe model was 1 μm³, and the grid spacing inside and in the vicinity ofthe waveguides was 1 nm. Although actual experiments would in most casesuse tightly focused light, rather than the plane waves modeled here, thedimensions of the waveguides modeled are small enough compared with thewavelength of light to make the plane wave approximation accurate enoughto estimate the intensity distribution within the waveguides. Modelswere run for waveguide diameters between 30 and 100 nm.

FIG. 11 shows the calculated intensity distribution for a 50 nm diameterwaveguide. The intensity falls off quickly with increasing propagationinto the waveguide, as expected for light that is well below the cut-offfrequency for the guide. The intensity distribution is relativelyconstant across the waveguide, as demonstrated in FIG. 11, and,therefore, one can estimate the effective illumination volume byplotting the intensity as a function of propagation distance. Theability of fluorescent photons to couple out of the waveguide will alsobe a strong function of distance from the entrance/exit pupil. Thiseffect has been assumed to follow a behavior that is similar to that ofthe excitation light propagation. However, this assumption fails to takeinto account the orientation of the emitting dipole, its exact laterallocation within the guide, or the different frequency of the emittedlight. The effective observation volume is described by the illuminationprofile multiplied by the collection efficiency profile. The effectiveobservation volume within a guide can, therefore, be approximated by thesquare of the intensity distribution. FIG. 12 shows the intensitysquared as a function of propagation distance for guides of variousdiameters decreases. As expected, the intensity decays faster as thewaveguide diameter decreases. Therefore, small diameter waveguides serveto decrease the effective observation volume both by physicallyconstraining the experiment in the lateral dimensions and by decreasingthe propagation distance of light into and out of the guide.

Example 3 Spectroscopy with Zero-Mode Waveguides

For fluorescence applications, one can define the size of effectiveobservation volume, V, as

$V = \frac{\int{{S(r)}{\mathbb{d}^{3}r}{\int{{S(r)}{\mathbb{d}^{3}r}}}}}{\int{{S^{2}(r)}{\mathbb{d}^{3}r}}}$where S(r) is the observation efficiency at the point r, and for thissystem S(r)=1²(r). FIG. 13 shows the effective volume of zero orderwaveguides in aluminum illuminated with 6×10¹⁴ Hz light as a function ofwaveguide diameter. Volumes are on the order of 50 zeptoliters (10⁻²¹liters). This compares to 0.5 femtoliters (10⁻¹⁵) for a typicaldiffraction-limited focal volume.

The effectiveness of the waveguides in confining the volume ofillumination was evaluated using fluorescence correlation spectroscopy(“FCS”). FCS involves illumination of a sample volume containing dyemolecules in solution. The diffusion of molecules into and out of theeffective observation volume leads to fluctuations in the number ofobserved molecules and hence to fluctuations in the fluorescence signal.These fluctuations occur on a time scale that is characterized by anaverage residence time of a molecule within the volume, τ_(D). Theautocorrelation of the fluorescence signal, G(τ), is given by

${{G(\tau)} = \frac{\left\langle {\delta\;{F(t)}\delta\;{F\left( {t + r} \right)}} \right\rangle}{\left\langle {F(t)} \right\rangle^{2}}},$where F(t) is the fluorescence signal at time t and δF(t) is thedeviation in fluorescence from the mean. G(0) is inversely proportionalto the average number of molecules in the volume and the half-max ofG(τ) occurs at the typical residence time of molecules diffusing in thevolume. For a known concentration of dye in solution, the average numberof molecules observed gives a useful estimate of the effectiveobservation volume and is critical for determining the expectedbackground from freely diffusing species in studies of single enzymemolecules in the presence of fluorescent ligands. The average residencetime of diffusing dye molecules, and the overall shape of G(τ), can becombined with theoretical calculations to give an understanding of theshape of the effective observation volume. The residence time is alsorelevant to the temporal resolution of studies of enzymatic dynamics.Reactions that produce fluorescent product or intermediates may bedistinguishable from background fluorescence fluctuations if therelevant reaction rates are longer than the typical diffusion time.

Arrays of waveguides were illuminated from the glass side with 488 nmcircularly polarized light from an argon ion laser using a 60× waterimmersion microscope objective (UPlanApo Olympus, Melville, N.Y.,NA=1.2). Fluorescence was collected by the same objective, passedthrough a dichroic mirror (dichroic long-pass 500, Chroma TechnologyCorp., Brattleboro, Vt.) and two emission filters (575/150 and 580/150)to block reflected laser light and coupled into a 50 μm optical fiber(OZ Optics Ltd., Corp., Ontario, Canada). A 50/50 fiber splitter wasused to send the signal to two avalanche photodiodes (“APD”s) (PerkinElmer Optoelectronics, Fremont, Calif.) for cross-correlation.Cross-correlation was necessary to remove artifacts from after-pulsingat short times in individual APDs. Cross-correlation is similar toauto-correlation and yields the same information, except that G(τ) isnow given by

${G(\tau)} = \frac{\left\langle {\delta\;{F_{1}(t)}\delta\;{F_{2}\left( {t + \tau} \right)}} \right\rangle}{\left\langle {F_{1}(t)} \right\rangle\left\langle {F_{2}(t)} \right\rangle}$where subscripts indicate signals measured at different APDs.

Waveguide arrays were exposed to oxygen plasma for one minute and werepre-treated with a solution of heparin (50 μg/ml) in HPLC grade water onthe aluminum side to prevent sticking of dye to the glass and metalsurfaces. FCS was then performed on arrays with a 1 μM solution of thedye Bodipy 515 pyrophosphate (see FIG. 14) in HPLC grade water with 50μg/ml of heparin. Molecules can diffuse into and out of the waveguideonly through the top of the waveguide and the gradient of lightintensity inside the waveguide is essentially one dimensional, asdiscussed above. Therefore, one would expect G(τ) to be dominated by thedecay in 5 illumination intensity and fluorescence output couplingefficiency with increasing distance from the entrance pupil, both ofwhich decay faster in smaller diameter waveguides.

Using the output from the finite-element simulations, theoretical FCScurves were generated for waveguides of various diameters and fit to thenormalized data curves. Despite the use of heparin and oxygen-plasmatreatment, the FCS curves displayed long-time tails attributable to somesticking of dye. This was accommodated for in the fitting function usingan additive exponential term such that the fitting function was of theform

${G(\tau)} = {{\frac{1}{N}{G_{diff}(\tau)}} + {Se}^{\frac{- t}{\tau_{s}}} + {offset}}$where G_(diff) is the numerically-derived diffusion component, S andτ_(s) are the sticking component amplitude and lifetime, and offset is asmall constant. FIG. 15 shows fits to waveguides of various diameters,with good agreement between the theoretical curves and data confirmingthe accuracy of the effective observation volume model. Theseexperiments, therefore, verify that zero-mode waveguides have beenconstructed and that they effectively confine the effective observationvolume to zeptoliter dimensions.

The confinement mechanism of the zero-mode waveguide can add usefulnessto many of the single-molecule spectroscopic techniques known in theart. In addition to FCS, and cross-correlation spectroscopy, thetechnique of dual-color cross-correlation spectroscopy is described in“Dual-color fluorescence cross-correlation spectroscopy formulticomponent diffusional analysis in solution” Schwille, P.,Meyer-Almes, F. J., Rigler R. Biophys. J. Vol. 72 No. 4: pp. 1878-1886April 1997, which is hereby incorporated by reference. This techniquecan be extended to function with two-photon excitation as described in“Two-photon fluorescence coincidence analysis: rapid measurements ofenzyme kinetics”, Heinz, K. G., Rarbach, M., Jahnz, M. and Schwille, P.,Biophys J, September 2002 pp. 1671-1681 Vol. 83, No. 3 which is herebyincorporated by reference. Both of these techniques can be enhanced bythe inclusion of the zero-mode waveguide as a volume limiting technique.

It will be seen by one skilled in the art of single-moleculespectroscopy and analysis that several different types of presentationsof the analyte will be useful. Analytes can diffuse freely in solutionor be immobilized in the illuminated region of the zero-mode waveguide.In cases where there is more than one analyte and these various analytesare expected to interact with one another, all of the permutations ofbound and diffusing analytes are possible. For two analytes, both can befreely diffusing, one or the other of them can be immobilized, or bothcan be immobilized (such is the case where more than one fluorescentlabel is attached to different residues of a single amino acid chain toobserve the folding kinetics of the protein). In the case of observationof enzymatic activity (see example 4 herein), it is useful to immobilizethe polymerase molecule to the device, while allowing the dNTP analogsbearing the fluorescent labels to diffuse freely. In a study of DNA-DNArenaturation kinetics from P. Schwille et al 1997 above it wasadvantageous to have both components of the analyte diffusing freely.Many useful configurations of labels and biological molecules areoutlined in “Fluorescence spectroscopy of single biomolecules”, S.Weiss, Science, Vol. 283, pp. 1676-1683, which is hereby incorporated byreference. In the present invention, the zero-mode waveguide iscontemplated in conjunction with all of these configurations to improvethe signal-to-noise, temporal resolution and tolerance to high ligandconcentration.

Example 4 Observation of Enzymatic Activity in Zero-mode Waveguides

SEQUENASE, a commercially available exonuclease-deficient mutant of T7DNA polymerase (USB Corporation, Cleveland, Ohio), was immobilized onthe bottom of a zero-mode waveguide by 15 minute incubation of thezero-mode waveguide structure with a 1:10 dilution of the commercialstock solution (13 U/μl in 20 mM KPO₄, pH 7.4, 1 mM DTT, 0.1 mM EDTA,50% glycerol) in glycerol enzyme dilution buffer (provided with theSEQUENASE enzyme, this buffer contains 20 mM Tris-HCl, pH 7.5, 2 mM DTT,0.1 mM EDTA, 50% glycerol). The waveguide was made of a 50 nm thickaluminum film on a clean fused silica coverslip (25×25 mm square (fromEsco Products, Oak Ridge, N.J.)) with an array of waveguides ofdifferent sizes. After immobilization, excess unbound enzyme was washedaway by extensive flushing with 1× pH 7.5 buffer (40 mM Tris-HCl, pH7.5, 10 MM MgCl₂, 10 mM NaCl). The reaction of DNA polymerization usingthe fluorophore coumarin-5-dCTP instead of dCTP was initiated byincubating the waveguide with a reaction mixture containing 3 ng/μlprimed M13 DNA, 5 mM dithiothreitol, 7.5 μM DATP, coumarin-5-dCTP, dGTP,dTTP, 6 ng/μl single-stranded DNA binding protein in 1×pH 7.5 buffer.Primed M13 DNA was provided by annealing 2 μg of M13mp18 DNA to (−40)M13 primer (2 pmol) in a 20 μl volume of 40 mM Tris-HCl, pH 8.0, 20 mMMgCl₂, and 50 mM NaCl by heating for 2 minutes at 65° C. and subsequentslow cooling to <35° C. over 30 minutes.

As shown in FIG. 16, the zero-mode waveguide with immobilized polymerasewas illuminated from the glass side with 488 nm circularly polarizedlight from an argon ion laser using a 60× water immersion microscopeobjective (UPlanApo, Olympus, Melville, N.Y., NA=1.2). Fluorescence wascollected by the same objective, passed through a dichroic mirror(dichroic long-pass 500, ChromaTechnology Corp., Brattleboro, Vt.) andtwo emission filters (575/150 and 580/150, Chroma Technology Corp.,Brattleboro, Vt.) to block reflected laser light and coupled into a 100μm optical fiber (OZ Optics Ltd.). This fiber was used to send thesignal to an avalanche photodiode (APD, Perkin Elmer Optoelectronics,Fremont, Calif.) for detection of the fluorescence photons. The signalfrom the APD was sent to a correlator card (Correlator.com, Bridgewater,N.J.) in a computer, where the time trace of fluorescence was recordedand stored, and the autocorrelation function of the signal wascalculated.

FIG. 17 shows the FCS curves from a waveguide before, during, and afterthe polymerization reaction. Before initiation of the polymerizationreaction (gray solid curve; in the presence of 7.5 μM coumarin-5-dCTP,but in the absence of DNA), the FCS curve only shows a decay originatingfrom the fast diffusion of the fluorescent coumarin-5-dCTP, analogous toFIG. 15 for the diffusion of Bodipy-515-PP inside zero-mode waveguides.After initiation of the polymerization reaction by addition of allingredients necessary to support efficient DNA synthesis, FCS curves aredominated by fluctuations originating from incorporation ofcoumarin-5-dCTP into DNA by the enzymatic activity of DNA polymerase,with a much longer time constant of ˜2 ms. This is because thecoumarin-5-dCTP is incorporated into DNA which in turn is bound to themolecule of DNA polymerase, and therefore the fluorophore spends a longtime in the confined volume of the zero-mode waveguide, continuouslyemitting fluorescence until it is photobleached. After completion of DNApolymerization (all of the single-stranded DNA has been extended intodouble-stranded DNA), the FCS curve returns to the shape that wasobtained before initiation of the polymerization reaction (gray dashedcurve).

FIG. 18 shows a time trace of fluorescence during the period ofpolymerization of coumarin-5-dCTP into M13 DNA. Distinct bursts offluorescence are visible, corresponding to incorporation of acoumarin-5-dCTP molecule into DNA, and subsequent photobleaching of thefluorophore. Traces, such as shown in FIGS. 17 and 18, can be used forcharacterization of the DNA polymerization process on a single moleculelevel.

For the analysis of this example, the concentration of fluorophore(coumarin-5-dCTP) is fairly high, at 7.5 μM. The fact that singlemolecule enzymatic activity can be observed inside the waveguidedemonstrates that the zero-mode waveguide of the present inventionprovides a confined volume to enable such analysis. In unconfinedvolumes, the number of fluorophores would be far too high to permit theobservation of enzymatic turnovers of DNA polymerase. For example, in adiffraction-limited excitation volume of 0.2 fl, such as provided byfocusing laser light with a high numerical aperture objective lens, aconcentration of 7.5 μM corresponds to an average of ca. 900fluorophores simultaneously present inside the volume.

Example 5 Use of a Superstructure to Enable Use of Many Samples on aSingle Chip

The zero mode waveguide devices are very small and use a minute fractionof the available surface area of a moderate-sized chip. As seen in FIG.19, it is possible to replicate the pattern of holes indicated generallyat 1910 in the aluminum film 1915 many times over on a 25 mm squarefused silica chip. It is desirable to be able to apply each of thesedevices with a separate sample to increase the number of experimentsthat can be conducted with a single chip. A silicone(polydimethylsiloxane) superstructure such as silicone rubber gaskets1920 is applied to the aluminized surface after the fabrication of thezero-mode waveguides in the metal film. The silicone structure 1920contains an array of holes as seen in a perspective view of FIG. 19 at2010 coincident with the centers of the zero-mode waveguide devices sothat the union of these two forms wells into which a fluid sample can beplaced while preventing contamination of the neighboring zero-modewaveguide devices. Such devices are commercially available from GraceBiolabs, Inc. in Bend, Oreg. Through the use of this method a singlechip can be employed with dozens of separate samples. While silicone hasbeen used for this purpose, the present invention contemplates anysuitable material and means of attachment for the use of asuperstructure to isolate neighboring zero-mode waveguide devices.

Example 6 Use of a Superstructure to Enable Multiple Analyses of aSingle Sample

As with the previous example, a silicone superstructure 2110 is appliedto the aluminized surface 2120. The silicone superstructure 2110 ispatterned with grooves 2130 and rectangular recesses 2140 that whenmated with the aluminized surface create fluid channels and cavitiesrespectively. Techniques for creating microfluidic structures forapplication in this way are known in the art and are discussed intechnical papers such as “fabrication of a configurable, single-usemicrofluidic device”, McDonald, J. C., Metally, S. J. and Whitesides, G.M., Analytical Chemistry 73 (23): 5645-5650 December 1 (2001), which ishereby incorporated by reference.

FIG. 22 is a plan view of the structures. The placement of therectangular recesses 2140 in the silicone superstructure is chosen tocoincide with the locations of zero-mode waveguide devices 2150 on thefused silica chip. The fluid channels 2130 can be used simply to conveyliquid to the several cavities where different analyses will take place.For example, each cavity can be pre-loaded with different reagents priorto mating the superstructure with the chip so that each cavity enables adistinct experiment on the sample. Alternatively the fluid channels canbe employed to perform pre-processing of the sample before it arrives inthe cavity. For example, capillary electophoresis can be performed tofractionate the sample into components before delivering thesecomponents to separate cavities containing zero-mode waveguide devicesfor further analysis of the sample. Zero-mode waveguides such aswaveguide 2310 can also be placed to coincide with a channel 2320 asseen in FIG. 23, so as to allow analysis of material as it passesthrough the channel. Air vents 2230 can optionally be used in caseswhere trapped gas prevents the entry of fluid into the recesses or fluidchannels. These air vents can also serve as conduits to allow theintroduction of other materials to the cavities after the superstructurehas been mated to the fused silica chip. These microfluidic channels andfluid cavities can also facilitate the use of much smaller quantities ofsample than can be conveniently managed using hand-pipetting methods.While silicone has been used for this purpose, the present inventioncontemplates any suitable material and means of attachment for the useof a superstructure to provide microfluidic channels and fluid cavitiesin conjunction with zero-mode waveguides to allow multiple analyses inparallel on a single sample.

CONCLUSION

In the present invention, the region of observation is internal to thewaveguide, as opposed to prior methods in which the region ofobservation is external to the sub-wavelength aperture. By making use ofthe light internal to the waveguide, the present invention achieves goodlight efficiency.

The extreme confinement of the effective observation volume by thezero-mode waveguide of the present invention enables a proportionalincrease in the limiting concentration of analytes up to which singlemolecule detection is still possible. This is important for manyapplications involving the study of single molecules. Dorre, et al.,“Highly Efficient Single Molecule Detection in Microstructures,” J.Biotech. 86:225-36 (2001), which is hereby incorporated by reference. Asmany processes, particularly many biochemical reactions, occurefficiently only at concentrations much higher than the pico- ornanomolar concentration regime typically required for single moleculeanalysis in solution, zero-mode waveguides offer the potential to studythese processes at more appropriate concentrations, for example, underphysiologically relevant conditions. With the use of zero-modewaveguides, single molecule characterization is possible at much higherconcentrations, ranging into the micromolar regime, thus extending therange of biochemical reactions that can successfully be studied on asingle molecule level. Zero-mode waveguides, therefore, provide thefield of single molecule research with novel instrumentation so thathigher concentrations of analytes can be studied and higher backgroundcan be tolerated.

In addition to permitting the use of higher concentrations, thezero-mode waveguides of the present invention permit analysis of smallvolumes with the feature that signal fluctuations from diffusion ofanalytes occurs about 100 times more rapidly than by using a volumecreated by high numerical aperture objective lenses. As a result,enzymatic turnovers or chemical reactions can be more confidentlydistinguished from diffusion. The diffusional residence time of amolecule inside the waveguide is relevant to the temporal resolutioncapabilities of studies of enzymatic and chemical dynamics by setting alower limit of what time regime can be measured. Reactions that producefluorescent products or intermediates are distinguishable fromdiffusional background fluorescence fluctuations if the relevantreaction rates are longer than the typical diffusion time. Thus, one cananalyze faster processes than would be possible without using zero-modewaveguides.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method for analyzing an enzymatic reaction comprising: (a)providing a waveguide comprising: a substrate layer; a cladding layerdisposed upon the substrate layer; and a core comprising a hole disposedin the cladding layer, wherein the hole is dimensioned such thatelectromagnetic radiation entering the core provides an effectiveillumination volume that is substantially internal to the core of saidwaveguide; (b) introducing an enzymatic reaction mixture comprising anucleic acid polymerizing enzyme, a target nucleic acid molecule, aprimer sequence, and a nucleotide analog into the core of the waveguide;and (c) directing excitation radiation at the core to illuminate theenzymatic reaction mixture in the effective illumination volume; and (d)analyzing the enzymatic reaction in the illumination volume.
 2. Themethod of claim 1, wherein the substrate layer is substantiallytransparent to electromagnetic energy.
 3. The method of claim 2, whereinthe electromagnetic radiation enters the core through the substratelayer.
 4. The method of claim 1, wherein the waveguide further comprisesa superstructure formed on top of the cladding layer having channels todirect the enzymatic reaction mixture into the core for analysis.
 5. Themethod of claim 1, wherein the nucleotide analog is fluorescentlylabeled.
 6. The method of claim 1, wherein the enzymatic reactionmixture comprises more than one type of nucleotide analog.
 7. The methodof claim 1, wherein at least one of the nucleic acid polymerizing enzymeand the target nucleic acid molecule is immobilized within the core. 8.The method of claim 1, wherein the hole comprises a diameter that isbetween 1 nm to 100 nm.
 9. The method of claim 1, wherein the holecomprises a diameter that is between 30 nm to 100 nm.
 10. A method foranalyzing an enzymatic reaction comprising: (a) providing an array ofwaveguides comprising: a transparent substrate layer; a cladding layerdisposed upon the substrate layer; and a plurality of cores, whereineach of the plurality of cores comprises a hole disposed in the claddinglayer and wherein the plurality of cores are configured to substantiallypreclude electromagnetic energy of a frequency less than a cutofffrequency entering the core from propagating longitudinally through saidwaveguides; (b) introducing an enzymatic reaction mixture comprising anucleic acid polymerizing enzyme, a target nucleic acid molecule, aprimer sequence, and a nucleotide analog into at least one of the coresof the waveguide; and (c) directing excitation radiation at the at leastone of the cores to illuminate the enzymatic reaction mixture; (d)analyzing the enzymatic reaction within the core.
 11. The method ofclaim 10, wherein the electromagnetic radiation enters said at least onecore through the substrate layer.
 12. The method of claim 10, whereinthe array further comprises a superstructure formed on top of thecladding layer, the surperstructure comprising one or more channels todirect the enzymatic reaction mixture into at least one of the cores foranalysis.
 13. The method of claim 10, wherein the nucleotide analog isfluorescently labeled.
 14. The method of claim 10, wherein the enzymaticreaction mixture comprises more than one type of nucleotide analog. 15.The method of claim 10, wherein at least one of the nucleic acidpolymerizing enzyme and the target nucleic acid molecule is immobilizedwithin the at least one of the cores.
 16. The method of claim 10,wherein the hole comprises a diameter that is between 1 nm to 100 nm.17. The method of claim 10, wherein the hole comprises a diameter thatis between 30 nm to 100 nm.